Q beta amplification.
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Gene Amplification↗
Biomedical subjects
C G Pritchard
Publications and source records attributed to C G Pritchard.
Amplified detection of viral nucleic acid at subattomole levels using Q beta replicase.
A sensitive, amplified assay for HIV-1 pol region RNA was developed using RNA probes which are replicated by the RNA-dependent RNA polymerase, Q beta replicase. A synthetic target RNA was hybridized in cell lysates prepared with guanidine thiocyanate with an RNA reporter probe and four deoxyoligonucleotide "capture" probes. The RNA reporter probe was a recombinant MDV RNA molecule generated by transcription from a cloned cDNA template. Capture probes are synthetic oligonucleotides that are complementary to the target nucleic acid and that bear 3' poly d(A) tails. The ternary hybrids (of target RNA with capture probe and reporter probe) were captured on oligo d(T)-derivatized paramagnetic particles by hybridization with the d(A) tails of the capture probes. Non-hybridized reporter probes were removed by washing and successively eluting and recapturing the ternary hybrids on fresh particles. After three cycles of elution and capture, the hybrids were eluted in a low ionic strength buffer and the MDV RNA reporter probes were amplified directly by Q beta replicase. Amplified product RNA was detected by fluorescence using propidium iodide. The assay detects one femtogram (600 molecules) of a synthetic target RNA containing the pol region of HIV-1. The complete assay takes about 2.5 hours.
Fluorescence↗
Quantitative assays based on the use of replicatable hybridization probes.
Amplifiable hybridization probes--molecules with a probe sequence embedded within the sequence of a replicatable RNA--will promote the development of sensitive clinical assays. To demonstrate their utility, we prepared a recombinant RNA that contained a 30-nucleotide-long probe complementary to a conserved region of the pol gene in human immunodeficiency virus type 1 (HIV-1) mRNA. Test samples were prepared, each containing a different number of HIV-1 transcripts that served as simulated HIV-1 mRNA targets. Hybridizations were carried out in a solution containing the chaotropic salt, guanidine thiocyanate. Probe-target hybrids were isolated by reversible target capture on paramagnetic particles. The probes were then released from their targets and amplified by incubation with the RNA-directed RNA polymerase, Q beta replicase (EC 2.7.7.48). The replicase copied the probes in an exponential manner: after each round of copying, the number of RNA molecules doubled. The amount of RNA synthesized in each reaction (approximately 50 ng) was sufficient to measure without using radioisotopes. Kinetic analysis of the reactions demonstrated that the number of HIV-1 targets originally present in each sample could be determined by measuring the time it took to synthesize a particular amount of RNA (the longer the synthesis took, the fewer the number of targets originally present). The results suggest that clinical assays involving replicatable hybridization probes will be simple, accurate, sensitive, and automatable.
Autoanalysis↗